The overall goal of our research is a better understanding of biological membrane structure and function, including lipid- protein interaction, protein-protein interaction and membrane protein folding. Development of new and more reliable biochemical and biophysical methods, especially fluorescence techniques, is of special concern. We are developing new fluroescence quenching techniques which probe hydrophobicity, the depth of protein penetration into membranes and the oligomeric state of membrane proteins. It is an important aim of this project to demonstrate the power of these methods which we believe will find general use in studies of membrane structure. Specifically, our goal is to understand the behavior of diphtheria toxin. Three steps in the entry of this toxin into cells are under study. First, how and why the toxin changes from a hydrophilic to hydrophobic state. Second, the nature of the conformation of the toxin once inserted into a membrane. Third, how the poisonous A subunit of the toxin is released into the cytoplasm. These studies should increase our knowledge of insertion of proteins into membranes and translocations across membranes. Also, these studies will help in the rational design of improved immunotoxins for therapeutic application. Finally, they should shed light of the behavior of related toxin proteins and viral fusion proteins. Eventually these investigations will be extended to such proteins.